No description available.

Thesis (Ph. D.)--University of Chicago, Department of Physics, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

The discovery of the existence of the cell membrane has led to a search for its organization on a molecular scale. The advent of artificial lipid bilayers and the development of electron microscopy in the 1930's provided direct visual evidence for the existence of the cell membrane and drove forward models of membrane structure based its known composition of proteins, lipids and carbohydrates. The fluid mosaic model of membrane structure, based on thermo- dynamics and newly developed protein structural studies of the time, placed integral globular membrane proteins within a fluid phospholipid bilayer. This model allowed for the association of proteins into groups and the possible mobility of proteins within the lipid bilayer. At the the same time fluorescence microscopy demonstrated movement of proteins in the plane of the lipid bilayer. Since then experimental techniques have been developed that show protein complexes of varying sizes do exist and so this gives us the opportunity to ask how receptor proteins fit into the molecular organization of the cell membrane. This thesis presents an investigation into how the epidermal growth factor receptor (EGFR) organizes in the cell membrane of colorectal carcinoma cells. First a new cell line for studying the receptor by stably expressing the epidermal growth factor receptor conjugated to enhanced green fluorescent protein (EGFR-eGFP) in SW620 cells was developed. This is an interest- ing cell line because it originates from a colonic adenocarcinoma that during the process of metastasis has lost the ability to express the EGFR. It therefore provided an environment for the expression of the fluorescent form of the receptor more in keeping with its natural environment. The technique of total internal reflection fluorescence (TIRF) microscopy was used to visualize the fluorescently tagged receptor in the cell membrane. This technique uses the principles of total internal reflection to excite fluorescence in molecules located only 100 nm into the cell. Because sources of fluorescence from outside the illuminated area are minimized individual fluorescent molecules can be imaged. The spots in the images, produced by the fluorophores, were detected using a single molecule detection and tracking algorithm. The intensities of these detected spots were analysed and compared with that from a single molecule of enhanced green fluorescent protein (eGFP). This gave an estimate of the number of receptors contained within each receptor complex. Before ligand binding most of the receptors were found to be located in complexes containing up to eight molecules and most frequently they were found in complexes of two molecules. Larger complexes of receptors were found to have formed after activation of the receptor by its ligand.

Total internal reflection fluorescence microscopy (TIRFM) has been widely applied for the study of biomolecules because of their ability to quantify biomolecules in a sample pretreatment and enrichment free manner, when compared with those costly, sample consuming and labor intensive conventional detection assay. Here, we have applied the TIRFM imaging system for the direct quantification and analysis of the biomarkers for the age-related diseases. Three research works on the quantification and study of biomarkers with the aid of TIRFM were herein described.

Renin is the initiator and rate-limiting factor of the renin-angiotensin system, a major mechanism of blood pressure regulation. The synthesis and secretion of this active circulatory enzyme is confined exclusively to the dense core granules of kidney juxtaglomerular (JG) cells where its precursor prorenin is packaged, cleaved to the active form and stored for release on a regulated pathway. Given its importance, surprisingly little is known about this process, in part due to the difficulty in culturing primary JG cells in vitro and the lack of reliable cell lines. The initial aim of the current work was to investigate renin-containing granule dynamics in live JG cells. To achieve this, I attempted to derive novel cell lines from triple transgenic mouse models comprising immortalised granulated or non-granulated JG cells. Due to the nature of JG cells in culture, the use of these cell lines to investigate granulation was not feasible; therefore the culture of primary JG cell culture was modified and enhanced to visualise granule dynamics in live, primary JG cells for the first time. By isolating cells using a Percoll gradient and plating them on fibronectin-coated dishes, rapid and full adhesion of JG cells was achieved, as well as prolonged expression of renin from 3 days to up to 8 days post-isolation. Using this protocol to isolate JG cells from RenGFP renin reporter mice and identifying granules using the acidotropic fluorophore Lysotracker, granule dynamics were investigated in primary JG cells. High resolution, rapid image acquisition was performed using widefield and total internal reflection microscopy, showing that dense core granules respond dynamically to the β-adrenergic agonist isoproterenol, a known renin secretory stimulus. Two different pools of granules of varying granule diameters and dynamic parameters were identified optically, suggesting that separate granule pools were being identified. Mice null for the Ren-1d gene lack renin storage granules in their JG cells, however granulation was restored in Ren1d-null mice carrying a transgene encompassing the human renin (hRen) locus. Therefore in order to investigate the relationship between renin expression and the amount of granulation in JG cells, mice expressing human renin were used. To dissect the granulation phenotype in detail, 2D electron micrographs were taken of JG cells, which were immunogold stained to confirm renin content, and reconstructed in 3D. Female hRen mice showed a significantly higher volume of granulation and an increased granule number compared to males, a finding consistent with the sexually dimorphic expression of the transgene, supporting the hypothesis that granulation in JG cells is dependent on the level of renin expression. The macula densa (MD) is a critical sensor of flow and salt content in the blood; through extensive tubulo-vascular crosstalk known as tubuloglomerular feedback (TGF), it releases key signalling factors stimulating and inhibiting renin synthesis and secretion from JG cells. Ren-1d-/- mice showed a hypercellular and columnar MD plaque, which was not restored by the introduction of the hRen transgene, indicating that TGF may be impaired in these mice. Using an isolated, perfused juxtaglomerular apparatus model it was shown that high salt- and increased flow-induced TGF functioned effectively in Ren1d-/- and huRen+/-Ren1d-/- mice, although animals on a Ren1d-/- background showed decreased sensitivity of glomerular tuft contraction and abnormal calcium signalling within macula densa cells. In conclusion, an appropriate in vitro model was developed for investigating granule dynamics in JG cells, using which granule motion was visualised and quantified for the first time in these cells. Although JG cell granulation is required for normal MD morphology, it was shown to not affect JGA function.

No description available.

The goal of this research was to study the polarization effect on the achievability of mode locked laser signal in accordance to polarization dependency. Polarization characterization was studied in prior to mode locking to determine semiconductor optical amplifiers (SOA) polarization dependency. It was found that per SOA manufacturer a variation of dependency was recognized. Two setups were suggested based on dissipative soliton resonance (DSR) schemes, the external DSR with regenerative feedback and the total internal reflection (TIR) DSR. The laser operated at 1530nm for multiple SOA manufacturers and was capable of generating ultrashort pulses with different durations as listed in Table (3.2.3) and Table (3.3.2). For the external mode-lock, the maximum achieved average output power of 3.40mW corresponding to ~77.15fJ of output pulse energy through continuous pumping by gain modulation of an SOA used as the active medium. While the TIR mode-lock the maximum achieved average output power for mode-locked signal of 5.25mW corresponding to ~23.73fJ of output pulse energy.It was concluded that the output pulse of the external DSR setup showed polarization dependency for two SOAs A and C and provided mode locked signals with pulse width ranging between 1200ps and 1000ps with repetition rate approximately of 35MHz and 101MHz respectively. The TIR DSR had a variation in results also to be polarization related. While SOA D did not provide mode-locked signal output. The least polarization dependent SOAs E and X did not provide distinctive stable mode-locked signal. While increased sensitivity towards polarization however resulted in stronger mode locked signal for SOAs A and C with FWHM between 200ps and 350ps and a repetition rate of approximately 20MHz. For future work, it is recommended to adjust the setup length in addition to fine tuning polarizer and filters to improve pulse stability and eliminate the noise effect.

In this dissertation fluorescence-based sensors for dissolved gases were developed for remote health monitoring applications including urine analysis. Detection of dissolved gases demonstrate diagnostic potential in body fluids, and indicate the metabolism of microorganisms driven by contaminated water. The emphasis of this research was on optimizing the sensitivity of fluorescence-based fluid sensing systems by configuring key design parameters towards cost reduction. A review of urine analysis indicated several methods for imaging, particle analysis, and detection of dissolved analytes. The review encourages readers to consider integrating sensing systems to provide additional context to results. An optical Dissolved Oxygen (DO) sensor was reproduced using phosphorescent organometallic dyes. The sensitivity of the DO sensor was experimentally optimized by employing Total Internal Reflection (TIR) of excitation light within the multilayered device by controlling the incident angle and sensitive film thickness. Novel 3D ray tracing-based computer models were developed based on the experimental results to explain the sensitivity enhancement mechanism of TIR. The path of light within the device and fluorescence generation sites were visualized and relative sensitivity was predicted. The model was validated by comparison with experimental results and expanded to predict the relative sensitivity of devices using different coupling strategies. This new optical model enables researchers to select an optimal coupling and detection scheme given their unique sensor design and application. A fluorescence based optofluidic sensor for Ammonia was redesigned based on experiment and simulation results. An optofluidic chip reader was produced to measure fluorescence sensors using low cost consumer electronics. The sensitivity of the ammonia sensor module has not been demonstrated; however, identified design challenges will be overcome in future efforts. As a result of this research, the cost of optofluidic sensing systems may be reduced towards enabling widely deployed remote monitoring networks for health and water quality. / Thesis / Doctor of Philosophy (PhD) / Dissolved gases have been detected in fluid samples as indicators of health and microbial activity by measuring changes in the intensity of fluorescence emitted from gas sensitive fluorescent dyes. These sensors can often be miniaturized and integrated to measure several parameters from a single platform. Several sensing platforms may be integrated into a continuous monitoring network. However, the cost of complete remote sensing networks prohibits the widespread deployment of these devices. The aim of this research was to improve the sensitivity of fluorescence-based sensors, reducing dependence on expensive detectors and light sources. The sensitivity of a fluorescence based dissolved oxygen sensor was optimized using Total Internal Reflection. A computer model was developed to identify important design parameters and their contributions to sensor performance. The model was validated by comparison with experimental measurements. Finally, an optical ammonia sensor is under development based on the dissolved oxygen experiments and model results.

Actin cytoskeleton reorganization plays essential roles in many cellular processes such as cell structure maintenance, cell motility, and force generation. Cytoskeletal drugs are small molecules that act on cytoskeletal components by either stabilizing or destabilizing them. Swinholide A is an actin-binding drug derived from the marine sponge. Swinholide A binds actin dimers as well as severs filaments. The main objective of this project is to determine how Swinholide A modulates actin filament assembly dynamics in the presence of macromolecular crowding. We utilize total internal reflection fluorescence (TIRF) microscopy imaging to directly visualize Swinholide A-mediated actin filament disassembly and severing. Filament disassembly and severing are evaluated by calculating actin filament lengths and length distribution controlled by Swinholide A. This study helps us better understand the fundamental mechanism by which Swinholide A affects actin assembly and disassembly dynamics. Further studies will allow for investigating new methods of treatment for a range of different diseases that have pathogenetically high levels of filamentous actin, such as cystic fibrosis, as well as a drug to combat the explosive expansion of cancers.